Cloning and Characterization of Lacritin in Monkey

Abstract

Abstract: : Purpose: Lacritin enhances unstimulated secretion by the lacrimal gland and is a ductal cell mitogen. Lacritin is also stimulator of corneal epithelial cells. It is expected to be an important factor in maintaining the surrounding ocular surface. Monkey would be a relevant animal model in which to study the role of lacritin in ophthalmic physiology and pathology. No cDNA cloning or functional analysis of monkey lacritin has been performed. Thus, the purpose of this study was to clone and characterize the monkey orthologue of lacritin. Methods: Total RNA was extracted from monkey lacrimal glands. RT–PCR and RACE were based on human lacritin (GenBank accession # NM_033277). Tissue specificity and relative amounts of mRNA in eye tissues were also assayed by RT–PCR. Sequencing of cDNA and expression of recombinant monkey lacritin with a His–tag in E. coli was performed using standard techniques. Results: Monkey lacritin cDNA contained 547 base pairs with 411 base pairs in an open reading frame encoding a protein of 137 amino acids. Monkey lacritin showed 89% amino acid homology with human lacritin; one amino acid was deleted. The predicted MW was 14.1 kDa, and the isoelectric point was 5.5. Purified, recombinant monkey lacritin with a calculated MW of 18.0 kDa (additional 3.9 kDa for His–tag) showed anomalous migration at 24.0 kDa on SDS–PAGE (confirmed by immunoblotting). This migration position is lower than that of human lacritin. Lacritin mRNA was highly expressed in monkey lacrimal gland and moderately expressed in corneal epithelium and conjunctiva. Conclusions: The high homology found between monkey and human lacritin provides rationale for use of monkey tissues to determine relevant functions for lacritin. The finding of lacritin expression in lacrimal gland, corneal epithelium and conjunctiva agrees with an expected a role of lacritin on ocular surfaces.

Keywords: lacrimal gland • gene/expression • protein purification and characterization